The study of biological systems and particularly the understanding of human disease is dependent on the ability to detect changes caused in biological systems by or in response to a disease. Such changes provide means of diagnosis and offer insights into the targets for therapeutic compounds such as vaccines and medicines. A wide range of biological molecules need to be measured quantitatively to understand disease processes including nucleic acids, proteins, steroids, sugars and lipids. In this context, the ability to quantitatively detect such biomolecules using mass spectrometers has provided considerable advances in their study and application to human and also to veterinary disease. The same advances have also occurred in environmental analysis and monitoring, and in food and beverage manufacturing. In particular the use of stable isotopes to provide synthetic quantitative references has been developed in isotope dilution mass spectrometry for monitoring of all classes of biomolecules. However, these methods have traditionally required an available synthetic standard, which is not always possible.
Recently, a range of chemical mass tags bearing heavy isotope substitutions have been developed to further improve the quantitative analysis of biomolecules by mass spectrometry. Depending on the tag design, members of tag sets are either isotopic having the same chemical structure but different absolute masses, or isobaric and isotopomeric, having both identical structure and absolute mass. Isotopic tags are typically used for quantification in MS mode whilst isobaric tags must be fragmented in MS/MS mode to release reporter fragments with a unique mass.
An early example of isotopic mass tags were the Isotope-Coded Affinity Tags (ICAT) (Gygi, S. P. et al., (1999) Nat Biotechnol, 17, 994-999). The ICAT reagents are a pair of mass tags bearing a differential incorporation of heavy isotopes in one (heavy) tag with no substitutions in the other (light) tag. Two samples are labelled with either the heavy or light tag and then mixed prior to analysis by LC-MS. A peptide present in both samples will give a pair of precursor ions with masses differing in proportion to the number of heavy isotope atomic substitutions.
The ICAT method also illustrates ‘sampling’ methods, which are useful as a way of reconciling the need to deal with small populations of peptides to reduce the complexity of the mass spectra generated while retaining sufficient information about the original sample to identify its components. The ‘isotope encoded affinity tags’ used in the ICAT procedure comprise a pair of biotin linker isotopes, which are reactive to thiols, for the capture peptides comprising cysteine. Typically 90 to 95% or proteins in a proteome will have at least one cysteine-containing peptide and typically cysteine-containing peptides represent about 1 in 10 peptides overall so analysis of cysteine-containing peptides greatly reduces sample complexity without losing significant information about the sample. Thus, in the ICAT method, a sample of protein from one source is reacted with a ‘light’ isotope biotin linker while a sample of protein from a second source is reacted with a ‘heavy’ isotope biotin linker, which is typically 4 to 8 Daltons heavier than the light isotope. The two samples are then pooled and cleaved with an endopeptidase. The biotinylated cysteine-containing peptides can then be isolated on avidinated beads for subsequent analysis by mass spectrometry. The two samples can be compared quantitatively: corresponding peptide pairs act as reciprocal standards allowing their ratios to be quantified. The ICAT sampling procedure produces a mixture of peptides that still accurately represents the source sample while being less complex than MudPIT, but large numbers of peptides are still isolated and their analysis by LC-MS/MS generates complex spectra. With 2 ICAT tags, the number of peptide ions in the mass spectrum is doubled compared to a label-free analysis.
Further examples of isotopic tags include the ICPL reagents that provide up to four different reagents, and with ICPL the number of peptide ions in the mass spectrum is quadrupled compared to a label-free analysis. For this reason, it is unlikely to be practical to develop very high levels of multiplexing with simple heavy isotope tag design.
Whilst isotopic tags allow quantification in proteomic studies and assist with experimental reproducibility, this is achieved at the cost of increasing the complexity of the mass spectrum. To overcome this limitation, and to take advantage of greater specificity of tandem mass spectrometry isobaric mass tags were developed. Since their introduction in 2000 (WO01/68664), isobaric mass tags have provided improved means of proteomic expression profiling by universal labelling of amines and other reactive functions in proteins and peptides prior to mixing and simultaneous analysis of multiple samples. Because the tags are isobaric, having the same mass, they do not increase the complexity of the mass spectrum since all precursors of the same peptide will appear at exactly the same point in the chromatographic separation and have the same aggregate mass. Only when the molecules are fragmented prior to tandem mass spectrometry are unique mass reporters released, thereby allowing the relative or absolute amount of the peptide present in each of the original samples to be determined.
WO01/68664 sets out the underlying principles of isobaric mass tags and provides specific examples of suitable tags wherein different specific atoms within the molecules are substituted with heavy isotope forms including 13C and 15N respectively. WO01/68664 further describes the use of offset masses to make multiple isobaric sets to increase the overall multiplexing rates available without unduly increasing the size of the individual tags.
WO02007/012849 describes further sets of isobaric mass tags including 3-[2-(2,6-Dimethyl-piperidin-1-yl)-acetylamino]-propanoic acid-(2,5-dioxo-pyrrolidine-1-yl)-ester (DMPip-βAla-OSu).
Recently, with dramatic improvements in mass accuracy and mass resolution enabled by high mass resolution mass spectrometers such as the Orbitrap (Hu, Q. et al., (2005) J Mass Spectrom, 40, 430-443 & Makarov, A. (2000) Anal Chem, 72, 1156-1162), Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometers (Marshall, A. G. et al., (1998) Mass Spectrom Rev, 17, 1-35) and high resolution Time-of-Flight (TOF) mass spectrometers (Andrews, G. L. et al., (2011) Anal Chem, 83, 5442-5446), it has become possible to resolve millidalton differences between ion mass-to-charge ratios. This high resolution capability has been exploited to increase multiplexing of Isobaric Tandem Mass Tags using heavy nucleon substitutions of 13C for 15N in the reporter region which results in 6.32 millidalton differences between the respective reporter fragments upon analysis by MS/MS (McAlister, G. C. et al., (2012) Anal Chem, 84, 7469-7478 & Werner, T. et al., (2012) Anal Chem, 84, 7188-7194). Similarly, it has been shown that metabolic labelling with lysine isotopes comprising millidalton mass differences can be resolved by high-resolution mass spectrometry enabling multiplexing and relative quantification of samples in yeast (Hebert, A. S. et al., (2013) Nat Methods, 10, 332-334).
Despite the significant benefits of previously disclosed isobaric mass tags, the multiplexing rate has been limited to 10-plex in commercial reagents to date. In addition, tags comprising very small mass differences would be useful because labelled ions that are related to each other, e.g. corresponding peptides from different samples, would cluster closely in the same ion envelope with very distinctive and unnatural isotope patterns that would be readily recognisable and which will be much less likely to interfere with the identification of other different peptides.
Hence, there still remains the need for sets of tags, where each tag differs from the others by millidalton mass differences, for labelling peptides and biomolecules with multiplexing rates greatly in excess of 10-fold.